Contact Us
800.227.0627

DNA Methylation Analysis Techniques in Epigenetics

 
Print Print
11/22/2013

DNA Methylation Analysis Techniques in Epigenetics

DNA Methylation Analysis Techniques in Epigenetics

By Klaus D. Linse

After sequencing the human genome scientists began to realize that just knowing the genetic information of an individual is not sufficient to understand its phenotype. The way a DNA sequence is translated into function does not directly depend on the sequence itself. It has now become clear that the interaction of genes with environmental factors plays a role as well. The new field of epigenetics attempts to integrate the different chemical interactions or languages that genomes and environment use to communicate with each other. Chemically, epigenetics can be described as structural adaptations of chromosomal regions that reflect silenced or activated states of genes in response of the epigenetic signaling network of a genome to environmental factors. DNA methylation, indicating a chemical epigenetic change or imprinting, is the most widely studied modification. The modification is found within CpG dinucleotides. DNA methylation is a biochemical process where a DNA base, usually cytosine, is enzymatically methylated at the 5-carbon position. This epigenetic modification is associated with gene regulation, and is of paramount importance to biological health and disease. Over the years a growing number of chemical modification at different amino-acid residues mostly located at histone tails have been identified as well. Examples are acetylation, methylation, phosphorylation, ubiquitination and sumoylation, and new types of modifications are still waiting to be discovered. Other epigenetically important features are factors like nuclear positioning, noncoding RNAs, and microRNAs, among others, which are also associated with gene regulation and chromatin structure. In biochemical and biological science the speed of discovery of novel findings goes hand in hand with the development of new techniques and/or scientific instrumentation. The past few years have witnessed the introduction of a whole host of new techniques that allow for the characterization of the epigenome. Specifically, the number of studies that attempt to characterize the epigenome of normal or altered genetic states has grown exponentially. The following tables outline a list of techniques and methods as well as current microarray platforms that have been developed for the study of the epigenome and their applications in biology and medicine.

Table 1: A List of techniques Used for Genome-Wide DNA Methylation Analyses
Method Principle Reference
RLGS
(restriction landmark genomic scanning)		 Methylation-sensitive restriction digestion and 2D electrophoresis Kawai, J. et al.;Methylation profiles of genomic DNA of mouse developmental brain detected by restriction landmark genomic scanning (RLGS) method, Nucleic Acids Res, 21, 5604-8, 1993.
MCA or MCAM
(restriction landmark genomic scanning)		 Methylation-sensitive restriction digestion, printed membranes and dot-blot analysis or microarray hybridization Estecio, M.R. et al.; High-throughput methylation profiling by MCA coupled to CpG island microarray, Genome Res, 17, 1529-36, 2007.Ibrahim, AE. et al.; MMASS: an optimized array based method for assessing CpG island methylation, Nucleic Acids Res, 34, 36, 2006.
DMH
(differential methylation hybridization)		 Methylation-sensitive restriction digestion and microarray hybridization Huang, T.H. et al.; Methylation profiling of CpG islands in human breast cancer cells, Hum Mol Genet,8,459-70, 1999.
CpG Island Array Amplicons, representing a pool of methylated CpG DNA derived from these samples, were used as hybridization probes in an array panel containing 1104 CpG island tags. Pearlly s. Yan, Martin R. Perry, Douglas E. Laux, Adam L. Asare, Charles W. Caldwell, and Tim Hui-Ming Huang; CpG Island Arrays: An Application toward Deciphering Epigenetic Signatures of Breast. Cancer Clin Cancer Res April 2000 6; 143.
AIMS
(amplification of inter-methylated sites)		 Methylation-sensitive restriction digestion, ID electrophoresis Frigola, J. et al.; Methylome profiling of cancer cells by amplification of inter-methylated sites (AIMS), Nucleic Acids Res, 30, e28, 2002.
MSO microarray
(methylation-specific oligonucleotide)		 Bisulfite conversion, PCR, bead array hybridization Gitan, R.S. et al.; Methylation-specific oligonucleotide microarray: a new potential for high-throughput methylation analysis, Genome Res, 12, 158- 64, 2002.
ChIP-on-chip Chromatin immunoprecipitation with antibodies against MBDs and microarray hybridization Ballestar, E. et al.; Methyl-CpG binding proteins identify novel sites of epigenetic inactivation in human cancer, Embo J, 22, 6335-45, 2003.
NotI digestion coupled to BAC array Methylation-sensitive restriction digestion and microarray hybridization Ching, T.T. et al.; Epigenome analyses using BAC microarrays identify evolutionary conservation of tissue-specific methylation of SHANK3, Nat Genet, 37, 645-51, 2005.
MeDIP-on-chip Isolation by 5-methylcytosine antibody and microarray hybridization.Immunocapture followed by DNA microarray analysis to generate methylation profiles of all human chromosomes at 80-kb resolution. Weber, M. et al.; Chromosome-wide and promoter specific analyses identify sites of differential DNA methylation in normal and transformed human cells, Nat Genet, 37, 853-62, 2005.
MClp-on-chip
(methyl- CpG immunoprecipitation)		 Isolation by MBD-Fc beads and microarray hybridization. Gebhard, C. et al.; Genome-wide profiling of CpG methylation identifies novel targets of aberrant hypermethylation in myeloid leukemia, Cancer Res, 66, 6118-28, 2006.
HELP assay
(Hpall tiny fragment Erichment by Ligation- mediated PCR)		 Methylation-sensitive restriction digestion plus microarray hybridization.Intragenomic profiling and intergenomic comparisons of cytosine methylation. Khulan, B. et al., Comparative isoschizomer profiling of cytosine methylation: the HELP assay, Genome Res, 16, 1046-55, 2006.
Methylation-specific bead arrays Bisulfite conversion, allele-specific primer extension, and bead array hybridization Bibikova, M. et al.; High-throughput DNA methylation profiling using universal bead arrays. Genome Res, 16, 383-93, 2006.
MSNP
(single nucleotide polymorphism chip-based method for profiling DNA methylation)		 Methylation-sensitive restriction digestion and SNP-chip hybridization Yuan, E. et al.;, A single nucleotide polymorphism chip-based method for combined genetic and epigenetic profiling: validation in decitabine therapy and tumor/normal comparisons, Cancer Res, 66, 3443-51 , 2006.
MMASS
(Combining array-based assays)		 Combinations of methylation-sensitive restriction digestions plus microarray hybridization Nielander, I. et al.; Combining array-based approaches for the identification of candidate tumor suppressor loci in mature lymphoid neoplasms, APMIS 115, 1107-34, 2007.
MIRA-Assisted Microarray Analysis (methylated-CpG island recovery assay) Isolation of methylated DNA by affinity to the MBD2/MBD3Ll complex plus microarray hybridization Rauch, T. et al.; MIRA-assisted microarray analysis, a new technology for the determination of DNA methylation patterns, identifies frequent methylation of homeo domain containing genes in lung cancer cells, Cancer Res, 66, 7939-47, 2006.
MSDK (Methylation- specific digital karyotyping) Methylation-sensitive restriction digestion plus SAGE Hu, M. et al.; Methylation-specific digital karyotyping, Nat Protoc, 1, 1621-36, 2006.
aPRIMES
(Array-based profiling of reference- independent methylation status)		 Differential restriction and competitive hybridization of methylated and unmethylated DNA Pfister, S. et al.; Array-based profiling of reference independent methylation status (aPRIMES) identifies frequent promoter methylation and consecutive downregulation of ZIC2 in pediatric medulloblastoma, Nucleic Acids Res, 35, e51, 2007.
Expression profiling after demethylation Treatment with demethylating agents and expression Microarray in cells with and without treatment Suzuki, H. et al.; A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer, Nat Genet, 31,141-9, 2002.
MTA
(methylation target array)		 In MTA, linker-ligated CpG island fragments were digested with methylation-sensitive endonucleases and amplified with flanking primers Chuan-Mu Chen, Hsiao-Ling Chen, Timothy H.-C. Hsiau, Andrew H.-A. Hsiau, Huidong Shi, Graham 1. R. Brock, Susan H. Wei, Charles W. Caldwell, Pearlly S. Yan, and Tim Hui-Ming Huang; Methylation Target Array for Rapid Analysis ofCpG Island Hypermethylation in Multiple Tissue Genomes. Am J Pathol. 2003 July; 163(1): 37-45.
Table 2: Current Microarray Platforms Used for Epigenomic Studies
Microarray Platform for Epigenomics Resolution Number of C Iones/Oligos* Coverage
BAC/PAC clones 100-200 kb Up to ~33,000 ~ Complete genome
CpG islands 100-1000 bp Up to ~ 12,000 CpG islands
Oligonucleotides      
Promoter 25-60 bp** 244,000;385,000; 4.6 million Promoter regions
CpG island 25-60 bp** 244,000; 385,000 CpG islands
Tiling 25-60 bp** Up to 45 million (set of 7 arrays) ~ Complete genome
CpG-dinucleotide specific 1 bp Up to 1536 Selected promoters
* Improved microarrays with higher resolution are constantly being developed, so the number of oligos on a single array increases as new platforms become available.
** This is the size of the oligonucleotide; the final resolution depends on the method used to enrich the DNA for methylated sequences or histone modifications.

CHROMATIN IMMUNOPRECIPITAION

CHROMATIN IMMUNOPRECIPITAION is a technique were intact nuclei are gently fixed to maintain the physical relationship of DNA-binding molecules to genomic DNA. The chromatin (DNA plus bound molecules) is sheared to small fragments and incubated with an antibody that selectively immuno-precipitates one of the bound molecules. The binding sites of the molecule (usually a protein) of interest become apparent from their enrichment in the immune-precipitated fraction of the genome.

Table 3: Early non-specific and differential gene methylation analysis methods
Early non-specific method Early differential methods
Restriction endonuclease digestion, isotope incorporation, and TLC Isoschizomer digestion and isotope incorporation
Polyclonal leporine antibody, radiolabeled DNA HpaII PCR
RP-HPLC Methylation-specificRLGS
HPLC, mass spectrometry AP-PCR
SssI methyltransferase tritium labeling AIMS
Monoclonal, isothiocyanate labeled fluorescent anti-5mC  

Legend: TLC, thin-layer chromatography; RP-HPLC, reverse phase high performance liquid chromatography; Anti-5mC, anti-5-methylcytosine; RLGS, restriction landmark genome scanning; AP-PCR, arbitrarily primed polymerase chain reaction; AIMS, amplification of intermethylated sites.

Table 4: Other Methods
Sodium Bisulfate Treatment Microarray Technologies
Ligation-mediated PCR Methyl-sensitive restriction enzymes; CGI library
Bisulfite sequencing Anti-methylcytosine immune precipitation; SMRT
MS-PCR Anti-methylcytosine immunoprecipitation; Promoter array
MS-SNuPE Methyl-binding protein precipitation; CGI library
MS-SSCA Methyl-binding protein precipitation; CGI library
MS-HRM Sodium bisulfate treatment; Oligonucleotides
Bisulfite treatment to create new restriction sites Sodium bisulfate treatment Illumina; beadchip

Legend: MS-PCR, methylation-specific polymerase chain reaction; MS-SNuPE, methylation-specific single nucleotide primer extension; MS-SSCA, methylation- specific single-strand conformation analysis; MS-HRM, methylation-specific high resolution melting; CGI, CpGislandmicroarray; SMRT,submegabase resolution tiling array; MeDIP,methylated DNA immunoprecipitation; MeCIP,methyl-CpG immunoprecipitation; Oligonucleotides, Whole genome oligonucleotide array.

Table 5: DNA methylation analyses by next-generation sequencing.
Method Genome coverage
Bisulfite sequencing Whole genome
MeDIP-seq1 anti-5mC Enriched Methylated DNA
MBDiGS2 Enriched Methylated DNA
MRE-seq3 Size selected fraction
MMSDK4 Representative genome tags

1 Sequencing of immunoprecipitated anti-5mC DNA

2 Methyl-binding protein precipitated sequencing.

3 Methyl-sensitive restriction enzyme sequencing.

4 Modified methylation-specific digital karyotyping.

Time line of DNA methylation analysis

Harrison & Parle-McDermott published a paper in 2011 describing the speed in the development of new techniques to study DNA methylation. The following figure illustrates this quite nicely.

Time line of DNA methylation analysis

Time line of DNA methylation analysis. Early techniques used in the 1980s allowed to measure the amount of 5-methylcytosine within a particular genome. Since then a variety of methods have been developed that allow for a more detailed study of the epigenome. These new type of methods or assays include the use of methylation-sensitive restriction enzymes, immunoprecipitation, bisulfite sequencing, usually in combination with the polymer chain reaction (PCR), the use of microarrays, reversed-phase high-performance liquid-chromatography (RP-HPLC), methylation-sensitive single nucleotide primer extension (MS-SnuPe), combined bisulfate restriction analysis (COBRA), arbitrarily primed PCR (APPCR), amplification of inter-methylated sites (AIMS), reduced representation bisulfite sequencing (RRBS), and finally next-generation sequencing, to name a few.

Future of Methylome Analysis

It is expected that next-generation sequencing approaches for DNA methylation analysis will dominate for a while. Newer sequencing technologies, such as single-molecule real-time sequencing (SMRT) are needed to directly detect all known DNA methylation reactions without the need for bisulfate treatment. As pointed out by Harrison and Parle-McDermott the major developments in the methodologies for profiling and fingerprinting the human methylome have followed a clear progression toward innovative sequencing techniques that allow for single-pair resolution. It is expected that as the technologies improve the cost of genome-wide sequencing will decrease. This will result in new waves of data and the need for better bioinformatics tools to allow for the accurate analysis of vast datasets in the coming years.

Reference

Weixing Feng, Zengchao Dong, Bo He and Kejun Wang; Analysis method of epigenetic DNA methylation to dynamically investigate the functional activity of transcription factors in gene expression. BMC Genomics 2012, 13:532 doi:10.1186/1471-2164-13-532.

Alan Harrison and Anne Parle-McDermott; DNA Methylation: A Timeline of Methods and Applications Front Genet. 2011; 2: 74.)

Lanlan Shena and Robert A. Waterland; Methods of DNA methylation analysis. Curr Opin Clin Nutr Metab Care 10:576–581. 2007 Lippincott Williams & Wilkins.